马甲子提取物部分改善AOM/DSS诱导的小鼠结肠炎相关结直肠癌：来自转录组学和肠道菌群分析的见解

摘要(中文译文)

马甲子（Paliurus ramosissimus，PR）是《中华本草》及地方药物志中记载的传统民间药用植物。本研究中，马甲子提取物（PRex）定义为由马甲子叶制备的乙醇提取物。马甲子叶传统上用于清热解毒、消肿止痛，这些传统用途与炎症相关病症密切相关。由于结肠炎相关结直肠癌（colitis-associated colorectal cancer，CAC）以慢性肠道炎症和黏膜损伤为特征，马甲子为在实验性CAC模型中评估PRex提供了民族药理学依据。本研究旨在评估PRex在AOM/DSS诱导的CAC小鼠模型中的保护作用，并探讨PRex相关表型变化是否与PPAR相关代谢通路及肠道菌群改变有关。建立AOM/DSS诱导的CAC小鼠模型。主要机制分析包括对照组、模型组和PRex 820 mg/kg组，同时以五味苦参和Aspirin作为药效学参考组进行表型评价。以白桦脂酸作为单一指标质控化合物，用于基于HPLC-ELSD的PRex批次质量控制。评估一般状况、疾病活动指数（disease activity index，DAI）、结肠长度、组织病理学变化及PCNA表达。采用RNA-seq和16S rRNA测序表征宿主转录组及肠道菌群变化。采用qRT-PCR和Western blotting进行初步分子评估，并分析外部GEO数据集，为候选基因表达及共表达模式提供背景证据。HPLC-ELSD分析显示，该批次PRex中白桦脂酸含量为63.6 mg/g。PRex与部分表型改善相关，包括结肠长度增加和PCNA阳性增殖活性降低。然而，与模型组相比，组织病理学总损伤评分仅呈非显著性下降。转录组分析鉴定出PRex相关的反向调控基因主要富集于代谢相关通路，包括PPAR信号通路、视黄醇代谢以及糖酵解/糖异生。16S rRNA测序显示，PRex处理与肠道菌群组成及PICRUSt2预测功能谱的改变相关。探索性比较提示宿主转录组通路与预测的微生物功能通路之间可能存在重叠。qRT-PCR初步支持PPARα、CD36、FABP4和PCK1的表达趋势，Western blotting提供了支持性半定量信息。PRex与AOM/DSS诱导的CAC小鼠部分表型改善相关，主要体现为结肠长度增加和PCNA阳性增殖活性降低，而组织病理学总损伤评分未显著降低。转录组分析、16S rRNA测序及初步分子评估提示，PRex干预与PPAR相关代谢通路及肠道菌群谱的改变相关。PPARα、CD36、FABP4和PCK1可能是与PRex治疗相关的候选分子。然而，这些发现应被解释为探索性和假设生成性的，仍需进一步的剂量-反应、毒理学、药代动力学及因果验证研究。

Abstract

Paliurus ramosissimus (PR) is a traditional folk medicinal plant recorded in Zhonghua Bencao (Chinese Materia Medica) and local medicinal records. In this study, Paliurus ramosissimus extract (PRex) was defined as an ethanol extract prepared from PR leaves. PR leaves have traditionally been used for heat-clearing and detoxification and for relieving swelling and pain. These traditional uses are closely related to inflammation-associated conditions. Because colitis-associated colorectal cancer (CAC) is characterized by chronic intestinal inflammation and mucosal injury, PR provides an ethnopharmacological rationale for evaluating PRex in an experimental CAC model. This study aimed to evaluate the protective effects of PRex in an AOM/DSS-induced CAC mouse model and to explore whether PRex-related phenotypic changes are associated with PPAR-related metabolic pathways and gut microbiota alterations. An AOM/DSS-induced CAC mouse model was established. The main mechanistic analysis included the Control, Model, and PRex 820 mg/kg groups, while Wuwei Kushen and Aspirin were used as pharmacodynamic reference groups for phenotypic evaluation. Betulinic acid was used as a single-marker quality-control compound for HPLC-ELSD-based batch quality control of PRex. General condition, disease activity index (DAI), colon length, histopathological changes, and PCNA expression were evaluated. RNA-seq and 16S rRNA sequencing were performed to characterize host transcriptomic and gut microbiota changes. qRT-PCR and Western blotting were used for preliminary molecular assessment, and external GEO datasets were analyzed to provide contextual evidence for candidate gene expression and co-expression patterns. HPLC-ELSD analysis showed that the betulinic acid content in this batch of PRex was 63.6 mg/g. PRex was associated with partial phenotypic improvement, including increased colon length and reduced PCNA-positive proliferative activity. However, the total histopathological injury score showed only a non-significant decrease compared with the Model group. Transcriptomic analysis identified PRex-associated opposite-direction genes enriched mainly in metabolism-related pathways, including the PPAR signaling pathway, retinol metabolism, and glycolysis/gluconeogenesis. 16S rRNA sequencing showed that PRex treatment was associated with shifts in gut microbiota composition and PICRUSt2-predicted functional profiles. Exploratory comparison suggested a possible overlap between host transcriptomic pathways and predicted microbial functional pathways. qRT-PCR preliminarily supported the expression trends of PPARα, CD36, FABP4, and PCK1, while Western blotting provided supportive semi-quantitative information. PRex was associated with partial phenotypic improvement in AOM/DSS-induced CAC mice, mainly reflected by increased colon length and reduced PCNA-positive proliferative activity, whereas the total histopathological injury score was not significantly reduced. Transcriptomic analysis, 16S rRNA sequencing, and preliminary molecular assessment suggested that PRex intervention was associated with changes in PPAR-related metabolic pathways and gut microbiota profiles. PPARα, CD36, FABP4, and PCK1 may represent candidate molecules associated with PRex treatment. However, these findings should be interpreted as exploratory and hypothesis-generating, and further dose-response, toxicological, pharmacokinetic, and causal validation studies are required.

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